The overall goal of this project is to gain further insight into local Ca2+ signaling in neurons. Local spatio-temporal differences in cytosolic (Ca2+), together with localized intracellular Ca2+ receptor molecules, provide one mechanism for selective modulation of diverse cellular functions by the single messenger Ca2+ in the same cell. Our recent confocal imaging of ryanodine receptor (RyR) Ca2+ release channels, endoplasmic reticulum (ER) Ca2+ pumps and mitochondria in cultured dissociated frog sympathetic ganglion neurons has indicated six specialized cellular sub-domains in these neurons: a peripheral ER-rich shell, an underlying mitochondria-rich shell, a central cytoplasm, a peripheral and a central perinuclear ER and the nucleus at the nuclear pole. Our recent video rate confocal fluorescent Ca2+ indicator (fluo-4) imaging in these neurons has revealed discrete sub-plasma membrane sites of preferential initiation of Ca2+ release activated either by caffeine or by a single action potential. The peripheral Ca2+ transients presumably occur via Ca2+ release from peripheral ER since they are eliminated by ER Ca2+ depletion. We now propose to address the following aims. (1) To characterize Ca2+ release, Ca2+ uptake and Ca2+ propagation in and between each of the identified sub-domains in these neurons, and to simulate our observations with a 6 interconnected sub-domain model of the neurons. (2) To study the basis for local peripheral sites of preferential Ca2+ release during single action potentials, and to determine whether these preferential release sites may also generate discrete local Ca2+ release events (Ca2+) "sparks"). (3) To determine the effects of transmitter-induced activation and other physiological perturbations in neurons in culture as well as in ganglia. We will combine high speed "band scan" (4 or 8 ms per band) and line scan (63 us per line) confocal fluorescent Ca2+ imaging of neurons in culture and in partially dissected fresh ganglia, electrophysiology, rapid extracellular perfusion, release of caged compounds, cytosolic injection of cDNA and/or proteins and histochemical study of live and fixed neurons. Our results will provide new insights regarding local Ca2+ signaling mechanisms that could be compromised in neuronal disease.